Fluorescent protein development 1) We have a study of the little known green-to-red photoconversion which occurs in the commonly used EGFP variant. Numerous reports dating back to the late 1990s have found that different forms of the green fluorescent protein can photoconvert into a red fluorescent species during intense irradiation. Initially it was reported that low oxygen conditions were required, but a similar phenomenon was noted in the presence of oxidizing agents. Our own studies have found that EGFP can undergo a green-to-red photoconversion in aerated samples lacking oxidizing agents, suggesting it is an inherent property of the protein. A survey of FP variants has found one which does not undergo this process. Comparison of the primary structures shows several differences and by performing single point mutagenesis at those positions the green-to-red photoconversion has been recovered. Now we are targeting the amino acid position for mutagenesis in EGFP of interest to both inhibit photoconversion as well as enhance this phenomenon. As second part of this project is to analyze the photobleaching behavior (loss in green fluorescence) in concert with redding effect. The effect of point mutants around the chromophore is being used to map the influence of various amino acids as well as the specific regions of influence. 2) We collaborate with Joy Zhao and Peter Schuck on their development of new fluorescence ultracentrifugation techniques. As a consequence, we have commenced a project to survey numerous fluorescent proteins to better define their oligomerization characteristics. Our surveys of the literature have suggested these characteristics have not been rigorously determined using the proper ultracentrifugation analysis. Our hypothesis is that aberrant behavior observed when these proteins are tagged to some proteins of interest may be due to oligomerization. 3) We have an ongoing project to develop improved red fluorescent proteins. Current variants display low fluorescence, slow maturation, and/or oligomerization. Biochemical analyses of wild type proteins coupled with site-directed mutagenesis has led to our discoveries of variants with decreased self-association, increased brightness, and faster maturation. Cell biology projects 1) We collaborate with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest is gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. Data have been collected on single expressed proteins using uninfected cells expressing PAFP tagged versions of the pUL37x1. Ongoing work is shifting to 2-color imaging to compare the localizations of multiple proteins of interest. 2) With Raul Rojas, a staff scientist in the lab of Lawrence Tabak, we have undertaken a project to image with PALM the localization of Golgi apparatus enzymes. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. Specifically, we are interested in whether enzymes involved in early steps in sugar modifications of proteins are located in early compartments of the Golgi and vice versa. We have performed preliminary experiments using a marker consisting of the localization domain of galactosyltransferase and the photoactivatable fluorescent protein, PAmCherry. Raul has just finished developing plasmids containing the chimeras with the full length enzymes. In addition, we are working to determine the centers of mass for the proteins of interest in nococazole induced Golgi fragments to give indications of their relative positions within Golgi mini-stacks Instrumentation and imaging development 1) A total internal reflection fluorescence (TIRF) microscope system has been built by Yan Fu on an Olympus IX70 microscope obtained through property transfer. This instrument is been designed to provide a more homogeneous illumination pattern compared with traditional through objective TIRF by illuminating the sample from all angles possible for a through objective configuration. This is achieved by rapidly scanning the excitation beam in a circular pattern around the periphery of the objective rear aperture using galvanometers. By doing so, any diffraction patterns or other aberrations often found in TIRF imaging are averaged by imaging from various angles. We have developed a method to image at multiple positions with the TIRF excitation zone which effectively allows optical sections at 20-50 nm increment through the 200-300 nm illumination region. 2) We collaborated with Andy York and Hari Shroff on development of a new fluorescence imaging technique, two-step fluorescence microscopy. It is similar to two-photon excitation microscopy except it utilizes much lower illumination intensities. In this application, we utilized the positive photoswitching properties of Padron to improve optical section and resolution. The technique relies on the sequential absorption of two photons of light. The first activates the fluorophore and the second photon excites the molecule to produce fluorescence, thus it is a non-linear (two-photon) technique and bestows many of the advantages of two-photon excitation microscopy on this method.